As the proximate pathologic cause of ischemic stroke, focal deficits of cerebral blood flow are central to understanding stroke evolution and developing therapies for its treatment. Over the several years of this project we performed serial perfusion MRI studies in ischemic stroke patients. We previously reported that reduction of the volume of ischemia by at least 30% within 2 hours from the start of tPA therapy predicted good clinical outcome. From a further analysis of serial perfusion scans, we developed the pharmacodynamic concept of reperfusion half-life as a measure of thrombolytic drug activity. We hypothesized that the probability of reperfusion relates to time according to an exponential decay function, analogous to plasma concentration half-life, and that reperfusion half-life following IV tPA therapy would indicate more rapid rates of reperfusion than those in the untreated condition of spontaneous reperfusion. In untreated patients (spontaneous reperfusion), a monoexponential decay function described the data well (R-squared = 0.95) with t = 29.1 hours. In tPA-treated patients, a biexponential decay function, with fast and slow components, was required to describe the data (R-squared = 0.99). The slow component was similar to that of the untreated condition; t=29.2 hours. The fast reperfusion component, attributable to tPA therapy, had t=0.7 hours. By approximately 3.5 hours after start of treatment, the effect of tPA on the probability of reperfusion was negligible. Our data show that modeling reperfusion as an exponential decay function distinguishes spontaneous from tPA-associated reperfusion and provides a measure of the speed and duration of thrombolytic activity. Having reached the milestone of over 1000 potentially treatable acute stroke patients (NIH Stroke Scale &#8805; 4 within initial MRI evaluation within 24 hours from stroke onset), we have undertaken the Lesion Evolution in Stroke and Ischemia On Neuroimaging (LESION) analysis. This unique, comprehensive dataset of acute stroke MRI affords unprecedented opportunities for robust statistical analysis of the incidence of ischemia and reperfusion as related to time from stroke onset. Among the results from ongoing and completed analyses, progress over the last year includes the following results. We may now quantify the unmet need and therapeutic opportunity for effective ischemic stroke therapy beyond 6 hours from stroke onset. Approximately 76% of patients presenting 6-12 hours from stroke onset have a perfusion defect; 47% of patients have a diffusion-perfusion mismatch, the penumbral pattern that is the target of reperfusion therapies. Logistic regression models predict that as many as 20% of ischemic stroke strokes presenting with NIH Stroke Scale &#8805; 4 with have an ischemic penumbra as late as 24 hours from stroke onset. Imaging based predictors of stroke outcome and response to therapy are necessary for the utility and validation of imaging biomarkers in drug development. Useful models are those that can distinguish patients destined for good outcomes versus poor outcomes, those who received effective therapy from those who did not, and treatment responders from non-responders. We are investigating several predictive models. These prediction models may be useful for the development, selection and use of acute therapies. We found that change in lesion volume from pre-treatment DWI to post-treatment FLAIR can discriminate between patients destined for good and poor outcomes when treated with effective acute stroke therapy, i.e., intravenous tPA. Thus, lesion volume change may be a useful marker of clinical response in the stroke therapy development. As we continue to evaluate quantitative, predicttive models of MRI parameters, we determined that qualitative evaluation of the diffusion-perfusion mismatch in thrombolytic eligible patients selects the same patients compared to retrospective quantitative mismatch measurements. Seventy patients from the LESION database who had MRI scans prior to standard thrombolytic therapy. Patient characteristics and thrombolytic outcomes (sex, age, NIHSS, mismatch volume, and mRS) did not differ for mismatch patients selected by qualitative versus quantitative methods. Qualitative mismatch selection among neurologists had a high sensitivity (0.82), specificity (0.80), accuracy (0.81) and positive predictive value (0.88) compared to core-lab quantitative measurements of mismatch volume. In 2004, we published the our discovery of an imaging marker of early blood brain barrier disruption (HARM - Hyperintense Acute Reperfusion Marker) in ischemic stroke and its relationship to reperfusion, hemorrhagic transformation, thrombolytic therapy, and worse clinical outcome. We have since confirmed that the localization of HARM on the gadolinium-enhanced FLAIR MRI is in the CSF space rather than in the parenchyma, proven an association of HARM with serum concentrations of matrix metalloproteinase-9, and demonstrated that HARM can be detected on immediate post-contrast, prior to treatment with tPA. The ability to detect of BBB disruption prior thrombolytic therapy may aid in the development of new therapies to protect the barrier and minimize the potential risks of thrombolytic therapy. To provide insight into the molecular mechanisms of acute ischemic cerebrovascular syndrome through gene expression profiling and pathway analysis we performed gene expression studies and pathway analysis in 39 patient with MRI confirmed acute ischemic cerebrovascular syndrome (AICS) and 25 nonstroke control subjects. A 9-gene profile was identified in the whole blood of ischemic stroke patients using gene expression profiling. Five of these 9 genes were identified in a previously published expression profiling study of stroke and are therefore likely biomarkers of stroke. Pathway analysis revealed toll-like receptor signaling as a highly significant canonical pathway present in the peripheral whole blood of patients with AICS. This study highlights the relevance of the innate immune system through toll-like receptor signaling as a mediator of response to ischemic stroke and supports the claim that gene expression profiling can be used to identify biomarkers of ischemic stroke. Further studies are needed to validate and refine these biomarkers for their diagnostic potential. Our study demonstrating the superiority of diffusion-weighted MRI to noncontrast CT for acute stroke diagnosis the key Class I study that formed the basis of an evidence-based guideline from American Academy of Neurology that DWI should be considered more useful than noncontrast CT for the diagnosis of acute ischemic stroke within 12 hours of symptom onset.